Insulin is an important target antigen in type 1 diabetes and a useful model antigen for analysis of mechanisms in antigen presentation and immune regulation. In this competing renewal application we propose experiments to further investigate antigen-processing mechanisms for presentation of insulin to T cells. We have identified T cells that recognize insulin presented by the nonclassical MHC class I molecule Qa-1. A major focus of the application is to characterize the function of Qa-1 and its role in the immune response to insulin. The specific aims are 1) to investigate the role of endosomal proteins in mediating disulfide reduction and antigen unfolding during antigen processing, 2) to analyze the peptide-binding behavior of the MHC class Tb molecule, Qa-1, 3) to characterize the determinant and investigate the pathway used by Qa-1-restricted T cells to recognize insulin, and 4) to investigate selection and function of insulin-specific, Qa-1-restricted T cells using TCR transgenic mice. This research is expected to broaden our general understanding of antigen processing and immune responses to insulin, as well as the function of Qa-1 and its human homolog, HLA-E.